chapter 2 review of literature -...

Review of Literature

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CHAPTER 2

REVIEW OF LITERATURE

This chapter deals with the scientific work carried out by researchers related

to composition of banana pulp and changes occurred in the pulp during ripening,

enzymatic clarification of fruit juices in general and of banana pulp in particular and

browning problems associated during clarification of fruit pulp and their inhibition.

The chapter is divided into 4 sections namely

2.1 Composition of ripened banana pulp

2.2 Changes during ripening of banana

2.3 Enzymatic extraction and clarification of fruit juices

2.4 Browning during clarification of fruit pulp and its

inhibition

2.1 Composition of Ripened Banana Pulp

Banana fruit is strongly recommended by nutritionists (Chandler, 1995), and

highly appreciated by consumers because of its flavour and sweetness. The

biochemical composition of banana fruits depends on the cultivar, abiotic factors such

as climate, cultivation method, soil type and storage conditions. The chemical

composition of ripened banana pulp studied by various researchers is as shown in

Table 2.1

According to data by various researchers presented in Table 2.1, the moisture

and total sugars content in banana pulp ranges from 63.8-76.0 per cent and 14.20-

20.18 per cent, respectively. Reducing sugars constitute the bulk of carbohydrate. The

reducing sugars present in ripe bananas are mainly fructose and glucose whereas non-

reducing sugar is sucrose.

Banana contains 2.93-7.00 per cent starch, 0.4-2.0 per cent fibre while pectin

content ranges from 0.34-1.10 per cent. Pectin stabilizes and gives a viscous body to

the pulp. Total carbohydrate in the pulp ranges from 21.8-27.2 per cent while 0.48-

1.50 per cent protein occurs. The major amino acids present in over ripen banana pulp

are aspartic acid, histidine, leucine and valine with total amino acid content of 3517

milligram per hundred gram (Askar, 1973).


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Table 2.1 Chemical Composition of Ripened Banana Pulp

Constituents Range References

Moisture (%) 63.8-76.0 1,2,3,4,5,7

Total sugars (%) 14.20-20.18 2

Reducing sugars (%) 4.10-18.89 2,7,8

Non reducing sugars (%) 0.00-16.08 2,7,8

Starch (%) 2.93-7.00 2,3

Fibre (%) 0.4-2.0 4,5,

Pectin (%) 0.34-1.10 2

Tannins (%) 0.007-0.050 6,7

Total carbohydrates (%) 21.8-27.2 4,5

Protein (%) 0.48-1.50 1,2,3,5

Fat (%) 0.20-0.47 1,2,3,5

Ash (%) 0.28-0.90 1

Potassium (mg %) 350-385 3,5

Magnesium (mg %) 30-42 3,5

Phosphorus (mg %) 22-36 1,4,5

Calcium (mg %) 8-17 3,4,5

Iron (mg %) 0.4-0.9 1,4,5

Vitamin C (mg %) 7-12 1,3,4,5,7

Niacin (mg %) 0.5-0.7 1,4,5

Vitamin B6 (mg %) 0.47-0.51 3,5

Riboflavin (mg %) 0.05-0.07 1,3,5

β-carotene (mg %) 0.050-0.078 1,4,5

Thiamine (mg %) 0.04-0.05 3,4,5

1. Select Committee on Nutrition and Human Needs, U.S Senate (1977)

2. Ockerman (1978)

3. Paul and Southgate (1978)

4. Gopalan et al, (1987)

5. CIQUAL-CNEVA (1993)

6. Kotecha et al, (1994)

7. Yousaf et al, (2006)

8. Ramesh Kumar et al, (2008)


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Fat content of banana pulp varies from 0.20-0.47 per cent (Ockerman, 1978).

The minerals present in the banana pulp include potassium, magnesium, phosphorus,

calcium and iron. Potassium constitutes about 39-58 per cent of total mineral matter.

Banana pulp has also been reported to contain ascorbic acid, niacin, vitamin B6,

riboflavin, β-carotene in good amount (Paul and Southgate, 1978; Gopalan et al.,

1987).

2.2 Changes during Ripening of Banana

Fruit ripening is a genetically programmed, highly coordinated process of

organ transformation from unripe to ripe stage, to yield an attractive edible fruit with

an optimum blend of colour, taste, aroma and texture. In banana, compositional

changes following harvest are important since banana is a climacteric fruit. Dramatic

changes in banana peel colour and pulp texture occur during the rise in respiration

during climacteric. In commercial trade, the ripening is initiated after transporting the

green banana to locations where the fruits are treated with ethylene. The research

work associated with changes during ripening of banana is as summarized below.

Kheng et al, (2012) determined the optimum harvest maturity and physico-

chemical quality of Rastali banana (Musa AAB Rastali) during fruit ripening. Rastali

banana fruit exhibited a climacteric rise with the peaks of both CO2 and ethylene

production occurring simultaneously at day 3 after ripening was initiated and declined

at day 5 when fruits entered the senescence stage. De-greening was observed in both

of the harvesting weeks i.e. 11 and 12 weeks after emergence of the first hand with

peel turned from green to yellow, tissue softening, and fruits became more acidic and

sweeter as ripening progressed. Sucrose, fructose and glucose were the main sugars

found while malic, citric and succinic acids were the main organic acids found in the

fruit. Rastali banana harvested at weeks 11 and 12 can be considered as commercial

harvest period when the fruits have developed good organoleptic and quality

attributes during ripening.

Soltani et al, (2011) investigated the changes occurred in physical and

mechanical properties of banana fruit (var. Cavendish) at different level of ripeness

during ripening in an airtight ware house with ethylene gas control system. Relation

between various stages of ripeness and these properties were determined and


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correlation coefficients were calculated. The colour of the fruit skin was measured as

L*, a* and b* in CIELAB system. The mechanical properties were extracted from

plotted force-deformation curve. A significant difference was found between the level

of ripeness and these properties except deformation. Results showed that changes in

L*, b* and C was similar, also variation of colour index (CI) was similar to a*. The

a* increased when banana fruits reached to a full-ripe stage. A positive correlation

was observed between a* and various stages of ripeness. As it was noted, an increase

in a* means a decrease in the degree of greenness. The firmness, rupture energy and

hardness decreased as banana fruit ripened. The firmness degraded from 75.1 N at

stage one to 27 N at stage seven. All measured physic-mechanical properties of

banana fruit except deformation had high correlation with stage of ripeness. Result of

deformation analysis showed no significant difference at various stages of ripeness.

The correlation between deformation and stage of ripeness was obtained as 0.2.

Kulkarni et al, (2010) studied the physico-chemical changes occurred during

artificial ripening of banana. Banana fruits harvested at 75–80% maturity were dip

treated with different concentrations of ethrel (250–1,000 ppm) solution for 5 min.

Ethrel at 500 ppm induced uniform ripening without impairing taste and flavour of

banana. Fruits treated with 500 ppm of ethrel ripened well in 6 days at 20±1 °C.

Changes in total soluble solids, acidity, total sugars and total carotenoids showed

increasing trends during ripening whereas fruit shear force values, pulp pH and total

chlorophyll in peel showed decreasing trends. Sensory quality of ethrel treated fully

ripe banana fruits was excellent with respect to external colour, taste, flavour and

overall quality.

Adeyemi and Oladiji (2009) studied the compositional changes in banana

(Musa spp) fruits during ripening. Banana fruits were collected, dried, ground and

ashed. The moisture content and mineral elements composition was determined as

ripening proceeds. The results showed that the nutritional composition of banana pulp

was diversely affected by ripening. The moisture content increased with ripening.

Changes in mineral composition varied and were not consistent with the stages of

ripeness. The magnesium content of the banana kept decreasing with ripening, while

increase in zinc and manganese reached a peak at the ripe stage and decreased

thereafter. Bananas were considered a good source of Mg in the diet, and the data


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obtained herein support these assertions. Zn and Mn were other minerals of

nutritional importance in bananas and this study has shown that their average values

are adequate to support its nutritive value at the various ripening stages. The result

obtained in this study showed that banana fruits at any ripening stage (unripe, ripe or

overripe) can be a potential source of mineral elements supplement in the diet

especially for Mg.

Duan et al, (2008) evaluated the changes in the pectin polysaccharide during

ripening of banana fruit. Pectin is one of the major components of the primary cellular

walls and middle lamella in plant tissues. In this study, water-soluble pectin (WSP)

and acid-soluble pectin (ASP) fractions were isolated from pulp tissues of banana

fruit at various ripening stages. Their monosaccharide compositions, glycosyl

linkages and molecular mass distributions were evaluated. As ripening progressed,

fruit firmness decreased rapidly, which was associated with the increase in the WSP

content and the decrease in the ASP content. Meanwhile, the molecular mass

distributions of WSP and ASP fractions exhibited a downshift tendency, indicating

the disassembly of pectin polysaccharides. Moreover, galactose and galacturonic acid

as the major monosaccharide compositions of pectin polysaccharides increased in

WSP fraction but decreased in ASP fraction during fruit softening. GC–MS analysis

further revealed that pectin polysaccharide had a 1,4- linked galactan/galacturonan

backbone with different types of branching and terminal linkages in WSP and ASP

fractions. During banana fruit ripening, the amount of 1,4-linked Galp residues of

ASP fraction decreased significantly whereas 1,3,6-linked Galp, 1,2-linked Manp and

4-linked Araf residues disappeared, which was related to depolymerization of pectin

polysaccharides. Overall, the study indicated that the modifications in polysaccharide

compositions and glycosyl linkages, reduced molecular mass distributions and

enhanced depolymerization of pectin fraction during banana ripening were

responsible for fruit softening.

Tadakittisarn et al, (2007) investigated the changes occurred in activity of

enzymes in bananas [Musa acuminate (AAA group) ‘Gros Michel’] associated with

the different ripening stages. Polygalacturonase (PG) and pectate lyase (PL) enzymes

from ripening stages 2-8 were extracted and partially purified by ammonium sulphate

fractionation. The results showed an increase in PG activity from 3.0±0.11unit/g fresh


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banana in the 2nd

stage to 4.89 ±0.39 unit/g fresh banana in the 6th

stage. Furthermore,

the PG activity decreased slightly to approximately 4.33±0.49 unit/g fresh banana as

the ripening stage increased (the 8th

stage). PL enzyme activity also depended on the

ripening stage of the banana. When the banana was ripening, the PL activity gradually

increased from 8.73 + 0.23 unit/g fresh banana in the 2nd

stage to 35.37 + 1.05 unit/g

fresh banana in the 8th Stage. When compared to the commercial pectinase enzyme

from Aspergillus aculentus (Pectinex Ultra SP-L, Novozymes A/S, Denmark), the

enzymes obtained from the banana demonstrated much lower activity. The PG and PL

activities from the commercial pectinase were 7966.46 and 1709 unit/ml,

respectively. The reducing sugar content also increased from 2.52 ±0.00 mg/g in the

2nd

stage to 118.5 ±0.00 mg/g in the 8th

stage of ripening.

Chen and Ramaswamy (2002) examined the kinetics of colour and texture

changes in ripening bananas as a function of storage temperature (10, 16, 22, 28 °C).

Colour was evaluated in terms of L, a and b values as well as the total colour

difference (ΔE) representing the residual deviations from the ripe stage. Puncture

force (PF) was used to evaluate the texture properties of banana. The results indicated

that the time dependence of L, ΔE and PF values followed a logistic model, while a

and b values were well described by a simple zero-order and fraction conversion

models, respectively. The Arrhenius equation adequately described the temperature

dependence of the reaction rate constants for both colour and texture parameters, from

which the activation energies and rate constant at reference temperature 15°C were

obtained. There were significant linear correlations between colour parameters (L, a,

b, ΔE) and texture parameter.

Prabha and Bhagyalakshmi (1998) investigated a comprehensive picture of

changes in carbohydrates, carbohydrate hydrolases, cell structure and texture in

banana fruit during ripening. Softening in the pulp during ripening was significant as

measured by the compression test on the pulp which decreased from 314 to 15N mm-2

from the raw to the ripe stage. The shear force decreased only to the extent of five

fold (from 133 to 27 N mm-2

indicating a much lesser degree of textural softening in

the peel. Starch (approx. 18%) had almost disappeared at the ripe stage. Total

hemicelluloses content lowered considerably from 2.4 to 0.9% during ripening

whereas pectin decreased from 1.1 to 0.8. There was no apparent change in cellulose.


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More than 80% of the radio activity of starch was incorporated into soluble sugars

viz. glucose, fructose and sucrose indicating active sugar inter conversions. The total

content of these soluble sugars increased from 1.8 to 19% with a concomitant

decrease in starch content during ripening. The various carbohydrate hydrolases viz.

polygalacturonase, pectin methyl esterase, xylanase, laminarinase, alpha-

mannosidase, beta-galactosidase, amylase, cellulase and hemicellulase registered a

general increase in their activities. Microscopically loss of cell wall integrity, cell

wall thinning, increased intercellular spaces, loosening of cells and disappearance of

starch granules were evident.

Wills et al, (1984) analyzed the proximate composition of `Cavendish’ banana

pulp at different maturity stages during ethylene induced ripening. Results showed

that the major changes in the pulp during ripening of the bananas were in the

carbohydrates. About 21 g/100 g of the pulp of the unripe fruit was starch, which

decreases continuously during ripening to reach 0.8 g/100 g in the fully ripe fruit

(stage 7= completely yellow with brown spots). Sugars were at low level in the unripe

fruit (1.3 g/100 g), but increase as a result of starch hydrolysis during ripening.

Sucrose was always the major sugar present; the major increase in sucrose occurs

early in the ripening process (stage 1= green to stage 3= green turning yellow) and

reaches a maximum (about 11 g/100 g) in firm ripe fruit (stage 7= fully yellow).

Fructose was always present at slightly lower levels than glucose. Both fructose and

glucose increased continuously during ripening and reach their maximum level in

fully ripe fruit (about 3 g/100 g and 4 g/100 g, respectively). Total carbohydrate

decreases by 5% during ripening, presumably because sugars are utilized in

respiration. The water content of the banana pulp increased during ripening from 72

g/100 g to 76 g/100 g. There was a decrease in dietary fibre early in the ripening

process (stage 1 to 3) and again when the fruits were fully ripe (stage 7). This change

was from 3.2 to 2.7 g/100 g as a result of hydrolysis of hemicelluloses and breakdown

of pectic substances.

Terra et al, (1983) observed that the accumulation of sucrose in banana fruit

preceded the increase of glucose and fructose during starch degradation. The

transformation of starch to sucrose was one of the possible mechanisms involved in


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the ripening of bananas. They proposed the following reaction mechanisms leading to

the formation of sucrose, the major sugar in banana.

Reaction (1) is catalyzed by phosphorylase enzyme.

Reaction (2) is catalyzed by UDP-glucose pyrophosphorylase.

Reaction (3) is catalyzed by sucrose-synthetase.

Lii et al, (1982) investigated changes during ripening of dessert bananas with

respect to physical and chemical properties of their starch and their content of

reducing sugars and sucrose. During ripening from maturity stage 1 starch content

decreases gradually whereas reducing sugar and sucrose content increases from stage

1 to fully ripened stage.

Wade and Bishop (1978) studied the changes in the lipid composition of

ripening banana fruits. The content of total lipid of banana fruit pulp tissue remained

constant during the climacteric rise induced by applied ethylene. The relative

proportions of neutral lipid, glycolipid and phospholipid did not change. However,

the fatty acid composition of the lipid did change during ripening. This change was

confined largely to the phospholipids fraction, in which there was an increase in the

proportion of linolenic acid and a decrease in the proportion of linoleic acid. The net

result was an increase in total unsaturation of the fatty acids in the phospholipids

fraction. The review of literature related to changes during ripening of banana is

summarized in Table 2.2

2.3 Enzymatic Extraction and Clarification of Fruit Juices

Over the last two decades, great advances have been made in the technology

of fruit juice processing. The advances have been towards the improvements in the

existing conventional processing procedures and equipments as well as introducing

new techniques to achieve maximum yield of juice with good quality. Enzymes

catalyses various reactions involved in the preparation of different food products.

(1) Starch (n) + Pi Glucose-1-P + Starch (n-1)

(2) Glucose-1-P + UTP UDP-glucose + PPi

(3) UDP-glucose + Fructose Sucrose + UDP


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Table 2.2 Review on Changes during Ripening of Banana

Sr.

No.

Author Year Important finding

1 Kheng et al 2012 Harvesting weeks i.e. 11 and 12 weeks after

emergence of the first hand could be considered

as a optimum harvesting period in Rastali banana

2 Soltani et al 2011 The surface colour of banana along with textural

properties was correlated with degree of ripeness

3 Kulkarni et al 2010 Artificially ripened bananas with ethrel were at

par in all qualities with naturally ripened bananas

4 Adeyemi and

Oladiji

2009 The nutritional composition of banana pulp was

diversely affected by ripening

5 Duan et al 2008 As ripening progressed, fruit firmness decreased

rapidly, which was associated with the increase

in the water soluble pectin content and the

decrease in the acid soluble pectin content

6 Tadakittisarn et

al

2007 Activity of polygalacturonase and pectate lyase

enzyme increased upto 6th

stage of ripening and

then slight decrease in 8th

stage.

7 Chen and

Ramaswamy

2002 Investigated the kinetics of colour and texture

changes in ripening bananas as a function of

storage temperature

8 Prabha and

Bhagyalakshmi

1998 Investigated a comprehensive picture of changes

in carbohydrates, carbohydrate hydrolases, cell

structure and texture in banana fruit during

ripening

9 Wills et al 1984 Major changes in the pulp during ripening of the

bananas were in the carbohydrates

10 Terra et al 1983 Accumulation of sucrose in banana fruit

preceded the increase of glucose and fructose

during starch degradation

11 Lii et al 1982 Investigated changes during ripening of dessert

bananas with respect to physical and chemical

properties of their starch and their content of

reducing sugars and sucrose

12 Wade and

Bishop

1978 The relative proportions of neutral lipid,

glycolipid and phospholipid did not change.

However, the fatty acid composition of the lipid

did change during ripening


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It is one of the important tools in modern food industry because while processing

many intermediate processes are simplified due to use of enzymes. Recently many

types of commercial enzyme preparations have found application as processing aid in

fruit juice processing industry too. A wide variety of enzymes are in use for different

purposes in fruit juice industry, among them pectinases are the most important in

extraction and clarification of fruit juices.

In this section, the research work associated with enzymatic extraction and

clarification of fruit juices has been summarized under following sub-topics:

2.3.1 Pectic substances and pectolytic enzymes in fruit juice processing

2.3.2 Extraction and clarification of fruit juices

2.3.3 Clarification of banana pulp

2.3.1 Pectic Substances and Pectolytic Enzymes in Fruit Juice

Processing

Pectic substances and pectolytic enzymes play an important role in fruit juice

processing. Only in the 1960s did the chemical nature of plant tissues become

apparent and with this knowledge, scientists began to use a greater range of enzymes

more efficiently. Pectinolytic enzymes are one of the important groups of enzymes

used in fruit processing industry. Several researchers have reported that

depectinization using pectinase could effectively clarify fruit juices. Primarily, these

enzymes are responsible for the degradation of the long and complex molecules in the

fruit pulp called pectin that occur as structural polysaccharides and responsible for

turbidity in pulp. Pectinases are now an integral part of fruit juice industries as well as

having various biotechnological applications.

Vaillant et al, (2001) worked on clarification study on six tropical fruit juices

(mango, pineapple, naranjilla, castillas blackberry, passion fruit, tangerine). He

observed that fruit juices contain colloids that are mainly polysaccharides (pectin,

cellulose, hemicelluloses and starch), protein and tannin. One of the major problems

encountered in preparation of fruit juices is cloudiness primarily due to the presence

of pectin. Usually tropical fruits are too pulpy and pertinacious to yield juice by

simple pressing. Pectin makes the fruit juices turbid and viscous and makes the

clarification process harder due to their fibre like molecular structure.


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Blanco et al, (1999) reviewed the classification of pectinases. Pectinases can

be divided into two main groups, namely pectin esterases (PE), which de-esterify

pectin by removing the methoxyl residues and depolymerases, which readily split the

main chain. Pectin esterases remove methoxy groups from high methoxy pectin to

give methanol and low methoxy pectin. The depolymerising enzymes can be

classified according to their preferred substrate, whether cleavage is random or

endwise, and if the enzyme acts by trans-elimination or hydrolysis.

Polygalacturonases (PG) cleave the glycosidic bonds by hydrolysis and pectate lyases

(PL) break the glycosidic bonds by β elimination. These enzymes can also be

classified according to whether they exhibit a preferential hydrolytic power against

pectin, pectic acid or oligogalacturonate as the substrate, and whether the mode of

action is random (endo-) or terminal (exo-). The classification of pectinases is shown

in Table 2.3.

Be Miller (1986) describe the classification of pectic substances. Chemically,

pectic substances are complex colloidal acid polysaccharides, with a backbone of

galacturonic acid residues linked by α (1-4) linkage. The side chains of the pectin

molecule consist of L-rhamnose, arabinose, galactose and xylose. The carboxyl

groups of galacturonic acid are partially esterified by methyl groups and partially or

completely neutralized by sodium, potassium or ammonium ions. Based on the type

of modifications of the backbone chain, pectic substances are classified into

protopectin, pectic acid, pectinic acid and pectin.

Baumann (1981) observed that temperature has a significant effect on the

activity of pectic enzymes. There was a close relationship between temperature and

time during enzyme treatment of fruit juices. As temperature increases, the rate of

pectin degradation was increases and the time of enzyme treatment decreases.

Rombouts and Pilnik (1978) reported that most of commercial pectic

enzyme preparations used in fruit juice extraction and clarification are of fungal

origin since these enzymes have a low pH optimum and other characteristics suited

for fruit juices. Pectic enzyme preparations mainly contain a mixture of pectolytic

enzymes like pectin methyl esterase (PE) and poly galacturonase (PG).They also

contain other non pectic enzyme activities such as cellulose, hemicelluloses, amylase,

esterase etc.


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Table 2.3 Classification of Pectinases

Group Enzyme Substrate Action

Polymethyl

galacturonases

(PMG)

Pectin Hydrolysis

Endo-

PMG

Random cleavage of α-

1,4glycosidic bonds

Exo-PMG Sequential cleavage of α-1,4

glycosidic bonds from the

non-reducing end

Polygalacturonase

(PG)

Pectic acid Hydrolysis

Endo-PG Random hydrolysis of α-

1,4glycosidic linkages

Exo-PG Sequential cleavage of α-

1,4glycosidic linkages from

the non-reducing end

Polymethyl

galacturonate

lyases (PMGL)

Pectin Trans-eliminative

cleavage

Endo-

PMGL



Exo-

PMGL

Sequential cleavage of α-


Polygalacturonate

lyases (PGL)

Pectic acid Trans-eliminative

Cleavage

Endo-

PGL



Exo-PGL Sequential cleavage of α-



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Neubeck (1975) reported the need for enzyme treatment varies with the type

of fruits and associated difficulties during juice preparation. Some fruits require the

enzyme treatment to expedite pressing for juice extraction. Other fruits may be

readily pressed without the need of adding enzymes but the cloudy pressed juices

would require enzyme treatment to improve their filtration to obtain clear juice. Such

differences are due to the wide variation in the physical and chemical composition of

different fruits particularly their pectic substances and the ratio of insoluble to soluble

pectin content. He also reported that the time required for flock formation during

clarification of apple juice was decreased by two folds for each 100C rise in

temperature in the range of 10-300C and by 1.5 fold at temp above 30

0C.

Endo (1965) studied the mechanism of enzymatic clarification of apple juice.

Cloudy apple juice contained soluble pectin with a small amount of insoluble pectin

bound to the suspended particles. Soluble pectin was found to act as a protective

colloid for other particles suspended in the cloudy juice and partial hydrolysis of this

pectin allowed other particles to flocculate and precipitate. The mechanism of

enzymatic clarification process involved 3 stages: solubilization of insoluble pectin,

decrease in viscosity of soluble pectin and finally flocculation of the suspended

particles. The review of literature related to this section can be summarized as shown

in Table 2.4

2.3.2 Extraction and Clarification of Fruit Juices

Bahramian et al, (2011) evaluated the effectiveness of enzymes pectinases

and cellulases in sugar extraction process from date fruits. Kabkab, a date cultivar

from Kerman province in Iran, which is industrially used for extraction of its sugar,

was selected for enzymatic extraction. Comparison of samples, pretreated by either

Pectinex®Smash XXL or Cellubrix® L with untreated date fruits, showed that

amount of both extracted sugar and clarity of juices thus produced, were affected by

enzymatic pre-treatment of fruits. Pre-treatment of fruits by each of the two enzymes

caused equally about 18% increase in the amount of extracted sugars, while using a

precisely determined mixture of two enzymes and a suitable condition, resulted in a

further increase of sugar to about 46%, in relation to untreated samples. Regarding the

clarity of the juice, the results indicated that increased sugar content of the extracts

positively affects the clarity of juices, with some exceptions.


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Table 2.4 Review on Pectic Substances and Pectolytic Enzymes in Fruit

Juice Processing

Sr.

No.


1 Vaillant et al 2001 Fruit juices contain colloids that are mainly

polysaccharides, protein and tannin. One of the

major problems encountered in preparation of fruit

juices is cloudiness primarily due to the presence

of pectin.

2 Blanco et al 1999 Reviewed the classification of pectinases.

3 Be Miller 1986 Described the classification of pectic substances.

4 Baumann 1981 There was a close relationship between

temperature and time during enzyme treatment of

fruit juices.

5 Rombouts and

Pilnik

1978 Most of commercial pectic enzyme preparations

used in fruit juice extraction and clarification are

of fungal origin since these enzymes have a low

pH optimum and other characteristics suited for

fruit juices.

6 Neubeck 1975 Enzyme treatment varies with the type of fruits

and associated difficulties during juice preparation

7 Endo 1965 Soluble pectin was found to act as a protective

colloid for other particles suspended in the cloudy

juice and partial hydrolysis of this pectin allowed

other particles to flocculate and precipitate.

Joshi et al, (2011) extracted the pectin methyl esterase from apple pomace

and evaluated its efficacy for extraction and clarification of plum, peach, pear and

apricot juice. The juice recovery of enzymatically treated pulps increased

significantly from 52-72% in plums, 38-63% in peach, 60-72% in pear and 50-80% in

apricot. Addition of pectinase significantly increased the total soluble solid (TSS),

titratable acidity and total sugar in the enzymatically treated juices. The pH, Brix acid

ratio and relative viscosity of extracted juices were decreased. The ascorbic acid

content remained unaffected with the increase in enzyme concentration. The overall

sensory evaluation of extracted juices using hedonic scale showed a significant

improvement in the colour and clarity scores. The flavour of extracted juices

remained unaffected. For extraction of juice 2.5% enzyme concentration was found to


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be best. However, for the clarification of apple and pear juice 1.0 and 0.5% enzyme

concentration gave the optimum results.

Karangwa et al, (2010) optimized the processing parameters for the

clarification of blended carrot-orange juice using Response Surface Methodology

(RSM) and to improve the carotene content. The blended carrot-orange juice was

treated with Pectinex Ultra SP-L enzyme at different concentrations (0.04- 0.12%),

pH (2.5-5), reaction temperature (40-60ºC) and time (70-110 min). The effect of these

independent variables on clarity, turbidity, and viscosity of the carrot-orange juice

was evaluated (coefficient of determination (R2) greater than 0.9). From the RSM

analysis, the optimum processing conditions were found as; 0.06% (w/v) enzyme

concentration, 3.6 pH, 49ºC temperature, 91 min reaction time. Clarified carrot-

orange juice thus obtained favourably improved the nutritional content and consumer

acceptance.

Shailza et al, (2009) evaluated the effect of four different clarifying

treatments viz. Sedimentation, Filtration, ‘Pectinase and Filtration’ and ‘Kaolin and

Pectinase’ for clarification of Kiwi and Peach fruits. Higher juice content was

obtained when the extraction was carried out in the presence of pectinase enzyme.

The recovery was higher when clarification of juice was carried out using the

combination of ‘Kaolin and Pectinase’ in both Kiwi (85.40%) and Peach (84.10%)

juice.

Vaidya et al, (2009) studied the enzymatic treatment for fruit juice extraction

from Kiwi fruit. Due to slimy pulp, extraction of pulp from Kiwi fruit was difficult.

To overcome this problem a combination of enzymes (pectinase 0.025g/kg + amylase

0.025g/kg + mash enzyme 0.05g / kg) were used to macerate the pulp (2h at 500C)

and thus facilitating the juice extraction. The treatment enhanced the juice recovery

(78.46%) compared to control (58.44%) and the treatment did not affect the TSS.

Titratable acidity, pH, reducing and total sugar of clarified juice. Also a drastic

decrease in the pectin content of the juice occurs. The outstanding feature of the juice

was its high acidity and high concentration of ascorbic acid which however decreased

by 21% after clarification.


22

Shah (2007) optimized the conditions for enzymatic extraction of Litchi pulp

with various concentration levels of hydrolytic enzymes viz. pectinase (0-

0.133%w/w), cellulase (0-0.266%w/w) and hemicellulase (0-0.20%w/w) for different

durations (30-150 min) at 45°C. Yield, clarity and TSS of juice were found to

increase and apparent viscosity was found to decrease significantly by enzymatic

treatment. The optimum conditions for enzymatic treatment of pulp obtained after a

double sided desirability function with the responses juice yield, clarity and TSS to be

maximized and viscosity to be minimized were 0.076% (w/w) pectinase, 0.138%

(w/w) cellulase, 0.107% (w/w) hemicellulase and incubation time of 106.5 min. The

predicted values for juice yield, clarity, viscosity and TSS under optimized conditions

were 77.19 %, 93.53%, 1.359mPa s and 19.68°brix which showed a good agreement

with the experimental values under the same set of conditions.

Sin et al, (2006) studied the enzymatic clarification of sapodilla juice.

Sapodilla juice was treated with pectinase enzyme at different incubation times (30–

120 min), temperature (30–50 °C) and enzyme concentration (0.03–0.10%). These

three factors were used as independent variables whose effects on turbidity, clarity,

viscosity and colour (L values) were evaluated. The results indicated that enzyme

concentration was the most important factor affecting the characteristics of the juice

as it exerted a significant influence on all the dependent variables. The recommended

enzyme clarification condition was 0.1% enzyme concentration at 40 °C for 120 min.

Sorrivas et al, (2006) studied the mechanisms governing the enzymatic

clarification of apple juice by electron microscopy techniques. Full ripe and unripe

apple juice samples were treated with commercial pectinase (Solvay 5XLHA) and

amylase (Rohalase HT) enzymes, respectively. Scanning electron microscopy studies

revealed that commercial amylolytic enzymes quickly reduced starch content in

unripe apple juice to undetectable values. It was also observed that after

pasteurization of this juice (900C, 5 min) all starch granules gelatinized. The effect of

pectic enzyme to destroy the protective pectin colloid was also detected with this

technique. It was speculated that the destruction of the weak pectin net by the action

of the specific enzyme caused particle aggregation, followed by the collapse of

aggregates, increasing the number of particles to less than 500 nm.


23

Lan Quin et al, (2005) investigated that the colour and cloud stability of

cloudy carrot juice were improved by enzymatic hydrolysis and addition of

hydrocolloids. Cellulytic and pectolytic enzyme preparations were used to prepare the

carrot juice and their optimum dosages were 1.6 g kg-1

and 0.035 g kg-1

, respectively.

Hydrocolloids including guar gum, pectin and flaxseed gum were each added to the

carrot juice and assessed for their ability to stabilize the carrot juice.

Singh et al, (2000) investigated the various physico-chemical changes during

enzymatic liquefaction of mango pulp (cv. Keitt). Pulps were treated at 400C for up to

2 h with a mixture of commercial enzymes, namely Pectinex Ultra SP-L, Celluclast

and Rapidase C PE at concentration of 1.5:6:20 v/v. Apparent viscosity of pulp and

serum reduced rapidly to 78 % and 93% respectively, in 30 min liquefaction. No

marked changes in apparent viscosity pulp samples and percentage cell wall

hydrolysis in the subsequent 60, 90 and 120 min were observed. The enzyme treated

pulp showed 83 % and 84 % serum yields during 30 and 120 min reaction time

respectively against 52% for untreated pulp. Slight increase in TSS (0Brix), acidity,

reducing and total sugars, and slight decrease in pH was found in the pulp as well as

serum fractions as the incubation continued. No marked change in colour

(yellowness) was observed in the enzyme-treated pulp, however, the corresponding

serum showed less yellowness and colour saturation indicating retention of yellow

pigments in the pulp fraction. About 9% loss in the total aroma components was

observed during the liquefaction process.

Brasil et al, (1995) prepared the clarified guava juice by treating the pulp with

600 ppm of pectic enzymes at 45°C for 120 min. The pulp so-treated was pressed to

give an average juice yield of 84.70%. The pressed juice was cloudy and pink in

colour but, after addition of fining agents and filtration, a clear juice with a light

yellow colour was obtained. This clear juice was preserved by the Hot-pack method.

During the extraction and clarification of the juice, some of the important physical

and chemical changes were followed by measuring changes in total soluble solids

(oBrix), acidity, viscosity, total phenolics content, colour, turbidity and ascorbic acid

retention.

Chang et al, (1995) investigated the efficacy of five commercial pectinases

for improvement of juice yield and quality from plums. Pectinases, to a varying


24

degree, improved the yield, color-assayed as release of anthocyanins, and clarity of

the juice. A significant increase in the effectiveness of pectinases was observed as the

concentration was increased from 0.01 to 0.60% v/w. However, at concentrations >

0.20% they tended to impart a bitter flavour in the juice. Among five pectinases,

Clarex L at 0.20% produced higher yield and a sediment-free clear juice.

Dawes et al, (1994) evaluated the effect of commercial fungal proteolytic

enzyme from Aspergillus niger in kiwifruit juice as a replacement for conventional

fining agents to produce a stable clarified juice. Reductions in detectable protein

levels of 73% and 82% were achieved using 500 mg/kg of enzyme and incubating at

60°C for 20 and 60 min respectively. Concentrates prepared from proteolytic enzyme-

treated juice had reduced browning and haze formation compared to a control,

without affecting ascorbic acid level. When stored at 20°C, proteolytic enzyme

treated concentrates (60 min) remained clear up to 90 days and had minimal haze

(A650 nm= 0.047) and browning (A420 nm= 0.93) after 6 months storage.

Sreenath and Santhanam (1992) found that a commercial pectinase from

Aspergillus niger containing various polysaccharases clarified the white grape juice to

an extent of 98-99% and also degraded the grape mash by 25-30%. This was achieved

by optimising the grape mash treatment with 0.048% of enzyme at 27-300C for 30

min without changing the mash pH. After pectinolytic juice clarification, both juice

viscosity and total phenols were reduced by 25% and 32% respectively. The review

of literature related to extraction and clarification of fruit juices can be summarized as

shown in Table 2.5

Table 2.5 Review on Extraction and Clarification of Fruit Juices

Sr.

No.


1 Bahramian et

al

2011 Pectinase and cellulose enzyme treatments to date

fruit yields juice with more sugar and clarity as

compared to untreated one.

2 Joshi et al 2011 Addition of pectinase significantly increased the

total soluble solid (TSS), titratable acidity and total

sugar in the enzymatically treated juices whereas the

pH, Brix acid ratio and relative viscosity of

extracted juices were decreased.

3 Karangwa et al 2010 Optimized the processing parameters for the


25

clarification of blended carrot-orange juice using

Response Surface Methodology

4 Shailza et al 2009 Evaluated the effect of four different clarifying

treatments viz. Sedimentation, Filtration, ‘Pectinase

and Filtration’ and ‘Kaolin and Pectinase’ for

clarification of Kiwi and Peach fruits

5 Vaidya et al 2009 Studied the enzymatic treatment for fruit juice

extraction from Kiwi fruit.

6 Shah 2007 The effect of enzyme treatment conditions was

studied on yield, clarity, apparent viscosity and total

soluble solids of litchi juice obtained from the pulp.

7 Sin et al 2006 Enzyme concentration was the most important

factor affecting the characteristics of the sapodilla

juice as it exerted a significant influence on all the

dependent variables.

8 Sorrivas et al 2006 Studied the effect of commercial pectinase and

amylase enzyme on full ripe and unripe apple juice,

respectively by using electron microscopy

techniques.

9 Lan Quin et al 2005 The colour and cloud stability of cloudy carrot juice

were improved by enzymatic hydrolysis and

addition of hydrocolloids.

10 Singh et al 2000 About 30 min enzyme treatment was sufficient to

bring down the pulp viscosity to a suitable level for

clarification. Prolonged incubation beyond this time

did not bring any additional desirable change;

instead it has an effect on total aroma content

11 Brasil et al 1995

Studied the physic-chemical changes during

enzymatic extraction and clarification of guava

juice.

12 Chang et al 1995 Pectinases at concentrations > 0.20% impart a bitter

flavor in the plum juice

13 Dawes et al

1994 A commercial fungal proteolytic enzyme from

Aspergillus niger was used in kiwifruit juice as a

replacement for conventional fining agents to

produce a stable clarified juice.

14 Sreenath and

Santhanam

1992 Commercial pectinase from Aspergillus niger

containing various polysaccharases could clarified

white grape juice to an extent of 98-99% and also

degraded the grape mash by 25-30%.


26

2.3.3 Clarification of Banana Pulp

Different researchers have used different commercial enzymes (especially

those with pectinolytic activities) in banana pulp processing. However most of the

researchers seem to have used only one stage i.e. ripened stage of banana pulp in their

study. In this study, pulp of three different ripening stages was used for clarification

study.

Cheirsilp and Umsakul (2008) prepared banana wine by treating the banana

must with pectinase and α-amylase to hydrolyze pectin and starch. The synergistic

activities of the enzymes enhanced hydrolysis of the complex carbohydrates. A

decrease of 55% in the viscosity and a 2.7-fold increase in the amount of extracted

juice were obtained after incubating with 0.05% (w/w) of pectinase at 400C for 2 h,

followed by treating with 0.05% (w/w) of a-amylase at 500C for 3 h. A 15 and 39%

increase in total soluble sugars and reducing sugars in extracted juice were achieved,

respectively. Enzyme-treated banana must was diluted with four volumes of water

and then fermented by yeast to produce banana wine. The pre-treatment of banana

with enzymes before wine fermentation resulted in a higher level of reducing sugars

than that of the control (non enzyme-treated banana wine) during fermentation. The

clarity of the enzyme-treated banana wine was also fourfold higher than that of the

control at 25 days of fermentation. The concentrations of total soluble solids, total

soluble sugars, and alcohol in the enzyme-treated banana wine and the control have

no significant differences.

Tadakittisarn et al, (2007) optimized the pectinase enzyme liquefaction of

banana ‘Gros Micheal’ pulp by response surface methodology (RSM). The effect of

pectinase enzyme concentrations (0-0.2%) and incubation times (60-300 min) on

juice yield (%), total soluble solids (TSS), recovery soluble solids (RSS), clarity

(%T670) and browning index (A420) of juice were studied using central composite

design of experiments. Results showed that pectinase enzyme concentration played an

important role that significantly (p ≤ 0.001) influenced most of dependent variables of

banana juice. The coefficient of determination (R2) of yield (%), recovery soluble

solids (RSS), clarity (%T670) and browning index (A420) were 0.907, 0.924, 0.804

and 0.793, respectively. The optimum condition for enzymatic extraction was 0.15%


27

of pectinase enzyme incubated for 120 min at 50°C. The yield was ≥ 62%, RSS ≥14,

%T670 ≥ 96% and A420 ≤ 0.1.

Lee et al, (2006) studied on optimization of conditions for the enzymatic

clarification process of banana juice using response surface methodology. Banana

juice was treated with pectinase at various enzyme concentrations (0.01– 0.1%),

temperatures (30–500C) and time (30–120 min) of treatment. The effect of these

enzyme treatments on filterability, clarity, turbidity and viscosity of the juice were

studied by employing a second order central composite design. The coefficient of

determination, R2 values for filterability, clarity, turbidity and viscosity were greater

than 0.900. Statistical analysis showed that filterability, clarity, viscosity and turbidity

were significantly (p<0.05) correlated to enzyme concentration, incubation

temperature and incubation time. Enzyme concentration was the most important

factor affecting the characteristics of the banana juice as it exerted a highly significant

influence (p<0.01) on all the dependent variables. An increase in time and/or

concentration of enzyme treatment was associated with an increase in filterability and

clarity, and decrease in turbidity and viscosity. Based on response surface and contour

plots, the optimum conditions for clarifying the banana juice were: 0.084% enzyme

concentration, incubation temperature of 43.20C and incubation time of 80min.

Shahadan and Abdullah (1995) determined the optimum conditions for

extraction of banana juice. RSM with a central composite design was used for

optimization. The effects of temperature (20-50ºC), pH (2.7-4.3) and enzyme

concentration (0.13-0.47%) on the yield of banana juice were studied after a 4h

reaction time. The optimal conditions for the enzymatic extraction of banana juice

were 0.42% enzyme at 35ºC with a pH of 3.4.

Yunchand et al, (1995) prepared the clarified banana juice by using

commercial enzyme pectinase and amyloglucosidase to increase juice yield. The

optimum concentration was 0.03% Pectinex Ultra SP-L or Rohapex TF with 0.02%

amyloglucosidase. Juice yield reached about 80% (base on pulp weight), when the

banana puree was preheated to 900C in steam blancher, followed by enzyme treatment

at 450C and maintained for 2 hrs. The effectiveness of enzyme on juice yield was

greatly depending on pre-heating the pulp and the stage of banana maturity.


28

Kotecha et al, (1994) carried out preliminary studies to carry out banana juice

extraction by using different levels of pectinase enzymes and different levels of

incubation periods at 28+ 20C. Based on these studies a 0.2% pectinase addition and a

4h incubation time were selected for obtaining the juice from pulp. The juice was

separated by centrifugation and the clear juice was used for preparation of juice.

Pheantaveerat and Anprung (1993) studied the application of commercially

available pectinases, cellulases and amylases for hydrolysis of ripe banana (Grade 7-

8) pulp. He observed that the synergistic activities of enzymes in increasing degree of

pulp hydrolysis expressed as percent decreased viscosity of juice obtained after the

pulp was incubated with 0.06% by weight of cellulases and with 0.05% by weight of

pectinases at 450C for 2 h. Under such condition, the clear juice yield of 73% (base on

pulp weight used) was obtained. Furthermore, amylases were not effective to the

above activity.

Koffi et al, (1991) conducted experiments to determine the effects of

commercial enzyme preparations on viscosity reduction and filterability of banana

juice and the effectiveness of various anti-browning treatments on clarified juice.

Two different combinations of pectinase, cellulase and hemicellulase were more

effective in reducing viscosity and improving filterability of both green and ripe

banana purees than a pectinase, galactomannanase or cellulase after incubation

periods of 3, 6 and 9 h. An alpha-amylase was not effective in reducing viscosity as

compared to the control, even of green banana puree high in starch.

Viquez et al, (1981) studied the pectinolytic enzyme treatments on banana

pulp to increase the yield, reduce the viscosity and clarify the juice. Clear juice yields

of between 55 and 60% (based on pulp weight used) are obtained from pulp incubated

at 45°C for 1 hr with 0.01% w/w of enzyme by subsequent centrifugation at 2900

maximal relative centrifugal force for 20 min. This corresponds to a yield of total and

reducing sugars present in the pulp of over 75%. Untreated control pulps yield less

than 5% of juice under these conditions. Hydraulic pressing of the pulps at 16 kg/cm2

gives similar juice yields to those obtained by centrifugation. The juice has an

excellent flavour and aroma and provides a possible use for the large quantities of


29

reject bananas available in producer countries. The research related to clarification of

banana pulp can be summarized as shown in Table 2.6

Table 2.6 Review on Clarification of Banana pulp

Sr.

No.


1 Cheirsilp and

Umsakul

2008 A decrease of 55% in the viscosity and a 2.7-fold

increase in the amount of extracted juice were

obtained after incubating banana pulp with

0.05% (w/w) of pectinase at 400C for 2 h,

followed by treating with 0.05% (w/w) of a-

amylase at 500C for 3 h.

2 Tadakittisarn et

al

2007 Dependent variables viz. yield, recovery soluble

solids, clarity and browning index are

significantly influenced by enzyme

concentration.

3 Lee et al 2006 An increase in time and/or concentration of

enzyme treatment was associated with an

increase in filterability and clarity, and decrease

in turbidity and viscosity.

4 Shahadan and

Abdullah

1995 The optimal conditions for the enzymatic

extraction of banana juice were 0.42% enzyme at

35ºC with a pH of 3.4.

5 Yunchalad et al 1995 The effectiveness of enzyme on juice yield was

greatly depending on pre-heating the pulp and the

stage of banana maturity.

6 Kotecha et al 1994 At 28±200C, 0.2% pectinase concentration and

4h incubation time yields maximum clarified

banana juice.

7 Pheantaveerat

and Anprung

1993 Amylases were not effective for the clarification

of pulp

8 Koffi et al 1991 Two different combinations of pectinase,

cellulase and hemicellulase were more effective

in reducing viscosity and improving filterability

of banana purees than alpha-amylase enzyme

9 Viquez et al 1981 Clear juice yields of between 55 and 60% were

obtained from pulp incubated at 45°C for 1 hr

with 0.01% w/w of enzyme concentration


30

2.4 Browning during Clarification of Fruit Pulp its Inhibition

Three general situations may cause browning reactions in fruit viz.

physiological changes associated with ripening, disorders associated with cold storage

or chilling injury and thirdly operations associated with harvesting and processing of

fruit where crushing, wounding or juice extraction occur. During preparation of

juices, once the fruit structure is disintegrated by crushing enzymes which are

normally associated with structural components would be brought into contact with

substrates and oxygen which stimulate some enzymatic and non enzymatic reactions.

These reactions may responsible to change in colour, flavour and appearance of the

juice (Pollard and Timberlake, 1971). Banana pulp is highly susceptible to

enzymatic browning during pulping. Browning reaction of banana fruit results from

the enzymatic oxidation of dopamine (3,4 -dihydroxyphenyl ethylamine) by

polyphenoloxidase (Griffiths, 1959) leading to the production of brown pigments.

Various techniques and mechanisms have been developed over the years for the

control of these undesirable enzyme activities. These techniques attempt to eliminate

one or more of the essential components (oxygen, enzyme, copper, or substrate) from

the reaction.

Samanta et al, (2010) evaluated the anti-browning (inhibition of polyphenol

oxidase activity) effect of cysteine (Cys) , ascorbic acid (AA), citric acid (CA),

sodium metabisulphite (SMB) alone or in combination, at three different pH (3.5, 4

and 4.5) in banana (Musa paradisiaca L. var. Kanthali), apple (Malus pumila Mill.

var. Ambri kashmiri), and mushroom (Agaricus bisporus). All the samples were

mixed with Cys (100, 200 and 300 mg/kg), AA (250, 500 and 1000 mg/kg), CA (250,

500 and 1000 mg/kg) and SMB (100, 200 and 300 mg/kg) to assess their effect on

PPO. PPO activity was analyzed spectrophotometrically at 420 nm. The most

effective PPO inhibitors were AA and SMB and in combination with CA and Cys in

all the samples tested. No significant differences were observed for PPO activity

among concentrations of Cys and CA when both anti-browning agents were used

alone or in combination and mixed with the samples.

Danyen et al, (2009) studied the interaction effects between ascorbic acid and

calcium chloride in minimizing browning of fresh-cut green banana slices. Dwarf


31

Cavendish banana having the highest browning potential was used for processing into

fresh-cut slices. The slices were treated with anti-browning agents, packed at 55%

vacuum level and stored at 100C. A 2 X 3 factorial treatment structure was used to

investigate the interaction effects between ascorbic acid and calcium chloride. At

three-day intervals, physicochemical parameters were investigated. The interaction

effect between ascorbic acid and calcium chloride for lightness and redness was

significant (P < 0.05); however, these colour parameters were mainly driven by the

main effect of ascorbic acid (P < 0.01). The interaction effect between ascorbic acid

and calcium chloride and the main effect of each chemical on firmness were highly

significant (P < 0.01). There was no interaction effect on yellowness (P > 0.05).

Browning and loss of firmness were promoted when 4% calcium chloride was used

singly and minimized when 2% ascorbic acid was added.

Chaisakdanugull et al, (2007) evaluated the effectiveness of pineapple juice

in enzymatic browning inhibition on the cut surface of banana slices. After storage of

banana slices at 150C for 3 days, pineapple juice showed browning inhibition to a

similar extent as 8 mM ascorbic acid but less than 4 mM sodium metabisulfite.

Fractionation of pineapple juice by a solid-phase C18 cartridge revealed that the

directly eluted fraction (DE fraction) inhibited banana polyphenol oxidase (PPO)

about 100% when compared to the control. The DE fraction also showed more

inhibitory effect than 8 mM ascorbic acid in enzymatic browning inhibition of banana

puree during storage at 50C for 24 h. Further identification of the DE fraction by

fractionation with ion exchange chromatography and confirmation using model

systems indicated that malic acid and citric acid play an important role in the

enzymatic browning inhibition of banana PPO.

Lee (2007) investigated the inhibitory effect of onion extract on banana

polyphenol oxidase activity during ripening of banana when stored at room

temperature for 10 days. The addition of the onion extract that had been heated at

100°C for 10 min exhibited a higher inhibitory effect on the banana polyphenol

oxidase activity during ripening of banana than that of the fresh onion extract. When

the onion extract that had been treated at a high temperature was added, the banana

polyphenol oxidase activity was markedly inhibited. It was found that heat treated


32

onion extract inhibited the banana polyphenol oxidase non-competitively. The

‘Millard Reaction Products’ (MRP) synthesized from arginine, cysteine, histidine and

lysine significantly inhibited banana polyphenol oxidase. The enzyme activity was

inhibited by addition of various anti-browning agents.

Zhang et al, (2004) investigated the characteristics of polyphenol oxidase

(PPO) of banana by spectrophotometer using catechol as substrate. The results

showed that the optimal temperature was 250C, pH 5.0. The PPO was inactive at 75

0C

for 5 minutes and the extinct condition of PPO was 20 s at 1000C. The PPO was

isoenzyme in banana. Effect of citric acid, ascorbic acid, sodium sulphite and cysteine

on PPO was also studied.

Ozoglu and Bayindirli (2002) studied the inhibition of enzymatic browning

in apple juice. Golden Delicious apple juice was subjected to enzymatic browning in

the presence of the selected anti-browning agents: ascorbic acid, isoascorbic acid, L-

cysteine, sorbic acid, benzoic acid, cinnamic acid and b-cyclodextrin. The relative

effectiveness of these anti-browning agents for inhibition of enzymatic browning in

apple juice was determined in terms of colour and enzyme activity measurements

with respect to time for approximately one day storage period at 25±10C. The most

effective agents were determined as L-cysteine, cinnamic acid and ascorbic acid.

Response surface methodology was used to evaluate the potency of the L-cysteine,

ascorbic acid and cinnamic acid combination for the control of enzymic browning.

The ascorbic acid, L-cysteine and cinnamic acid combination provided better results

than the individual compounds. The optimum combination was determined as 0.49

mM ascorbic acid, 0.42 mM L-cysteine and 0.05 mM cinnamic acid in the cloudy

apple juice stored for 2 h at 25±10C.

Almeida and Nogueira (1995) investigated the methods for the control of

polyphenol oxidase (PPO) activity in fruits and vegetables with the purpose of

reducing or eliminating the use of SO2 for this purpose. Interactions between the use

of ascorbic acid, citric acid, EDTA, sodium metabisulphite and heat treatment (70°C

for 2 min) in the control of PPO activity were studied in avocado (var. Fortuna),

banana (var. Nanica), apple (var. Ana, Fuji, Gala & Golden), pear (var. D'Agua),

peach (var. Real), potato (var. Bintje), eggplant (var. Super F100), mushroom


33

(Agaricus bisporus) and hearts-of-palm (Euterpe edulis Mart.). The results

demonstrated that PPO of avocado and eggplant was most resistant to inhibition by

the methods used. The least efficient method tested for the control of PPO was the

addition of EDTA, while the most efficient methods investigated included the use of

ascorbic acid, citric acid, sodium metabisulphite and heat treatment. The results

indicated that, with the exception of PPO from avocado, the most adequate alternative

method to substitute for the use of SO2 in the control of PPO was a combination of

ascorbic acid, citric acid and heat treatment.

Sims and Bates (1994) studied the processing and quality problems

associated with the production of clarified banana juice. For viscosity reduction

different groups of enzymes were used and to prevent browning several commonly

used methods were tested viz. heating whole, unpeeled bananas for 11min in 1000C

steam on a steam blancher belt (internal temperature reach to 850C); heating puree in

a small steam kettle with a constant stirring to 850C; addition of potassium

metabisulphite (100 mg/kg); addition of ascorbic acid (470 mg/ Kg). Results

indicated that the combination of pectinase, cellulase and hemicellulase was the most

effective of all the enzyme systems in reducing viscosity. Heating whole bananas or

purees to 80-900C for 1-2 minutes or the addition 100mg/ kg potassium

metabisulphite were effective in limiting browning.

Garda et al, (1985) developed a suitable and simple process for the

production of a puree, stable at ambient temperatures (28 to 300C), from optimum

ripened export-reject bananas in rural agro-industries. From this puree, different food

products such as a jellified snack ("bocadillo") can be manufactured. The effects of a

reducing agent (SO2) to prevent browning, acidification with citric acid, effect of

microbial inhibitors (sorbate and benzoate) and heat treatment on product quality

were studied. A combination of partial thermal enzyme inactivation in boiling water

(940C) for 7 minutes and immersion in 1% sodium bisulfite solution for 1.5 minutes

prevented browning. The addition of 1000 ppm potassium sorbate and citric acid to

pH 3.5 preserved the puree up to 4 weeks at 280C, retaining its original colour and

texture and an acceptable flavour. No significant microbial growth was detected.


34

Galeazzi and Sgarbieri (1981) reported that banana polyphenoloxidases

oxidize specifically o-diphenols. The Km values (Michaelis constant) were low for

dopamine, epinephrine, and norepinephrine. It was lower for L-dopa as compared

with D-dopa. The most effective inhibitor was ascorbic acid followed by cysteine and

then sodium metabisulphite. Diethyldithiocarbamate, at pH 7.0, was not as effective

as the above inhibitors. Ammonium ion and the oxidized β-nicotine adenine

dinucleotide acted as activators whereas Fe+2

, Fe+3, Al

+3, Ca

+2 and Zn

+2 showed

various degrees of inhibition. On the other hand, C+1

, Cu+2

, Mg+2

and Mn+2

did not

affect the enzyme activity. Mercaptoethanol (17mM) completely inactivated the

enzymes. On dialysis 30% of the activity was restored with regeneration of two

isozymes. The review of literature related to this section can be summarized as shown

in Table 2.7

Table 2.7 Review on Browning and its Inhibition during Clarification of Fruit

Pulp

Sr.

No.


1 Samanta et al 2010 In banana the most effective PPO inhibitors

were ascorbic acid and sodium

metabisulphite and the combination of citric

acid with cysteine

2 Danyen et al 2009 Dwarf Cavendish banana had the highest

browning potential. Browning and loss of

firmness were promoted when 4% calcium

chloride was used singly and minimized

when 2% ascorbic acid was added.

3 Chaisakdanugull et

al

2007 Pineapple juice could be used as a enzymatic

browning inhibitor in banana slices.

4 Lee 2007 Onion extract heated at 100°C for 10 min

exhibited a higher inhibitory effect on the

banana polyphenol oxidase activity during

ripening of banana

5 Zhang et al 2004 The PPO of banana was inactive at 750C for

5 minutes and the extinct condition of PPO

was 20 s at 1000C


35

6 Ozoglu and

Bayindirli

2002 Combination of ascorbic acid, L-cysteine

and cinnamic acid could effectively use for

inhibition of browning in cloudy apple juice

7 Almeida and

Nogueira

1995 In fruits and vegetables, the most adequate

alternative method to substitute for the use of

SO2 in the control of PPO was a combination

of ascorbic acid, citric acid and heat

treatment.

8 Sims and Bates 1994 Heating whole bananas or purees to 80-900C

for 1-2 minutes or the addition 100mg/ kg

potassium metabisulphite were effective in

limiting browning.

9 Garda et al 1985 In banana puree a combination of partial

thermal enzyme inactivation in boiling water

(940C) for 7 minutes and immersion in 1%

sodium bisulphite solution for 1.5 minutes

prevented browning

10 Galeazzi and

Sgarbieri

1981 The most effective inhibitor in banana was

ascorbic acid followed by cysteine and then

sodium metabisulphite.

chapter 2 review of literature -...

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